Study On Separation And Purification Of Human-like Collagen And The Functional Characterization Of Phosphatase Of Regenerating Liver-3

Collagen, the most abundant protein in the body, plays many aspects of physiologic function roles in the life. Recent research mainly has highlighted the importance of collagen in kinds of cancer therapy, including its key roles in cancer metastasis, angiogenesis, and cell growth, as well as its some poteintial utility in screening tests and as a therapeutic target. However, collagen has several advantages in the life, but different kinds of cancer (such as coloreatal cancer, breast cancer, liver cancer, lung cancer, cervical cancer, etc.) in human organisms are increasing year by year in modern life. The previous studies have shown that, these cancers have the common phosphatase (Phosphatase of regenerating liver-3, PRL-3) with overexpression, which promotes the rapid metastasis and exacerbation of cancer and leads to a rapid reduction and degrades of collagen in the human body. Thereby, the state of an illness change of patient will become worse, and the difficulty in the therapeutic processing will also be increased.Based on the importance of collagen and PRL-3 on human body, our study focused on the characterization of recombinant collagen and PRL-3 to provide a basis for further exploring the cancer therapy and drug development. To learn more changes of collagen and PRL-3 in cancer disease process is to provide a basis for therapy target of each patient according to the actual pathogenetic condition. Therefore, in this study, we worked on problems in the purification and separation of HLC to better apply in the clinical cancer patients.The pivotal problem of PRL-3 aberramt phosphorylationresulting in acquired or inherited human cancer diseases and immune deficiencies still remains unclear, so the investigationon PRL-3 phosphorylation is particularly important. This thesis identifies critical issues and newly available strategies for separating and purifying of collagen to improve the purity of HLC and studying on the functional chracteration of PRL-3 phosphoryaltion to further explore the relationship between recombinant HLC and PRL-3. The main contents of this thesis include the following aspects:1) HLC modified with polyol screens is separated and purified with cation-exchange chromatography. The optimal polyol-modified HLC was selected by testing different polyol additives, and some conditions of ion-exchange chromatography were carried on optimization. Polyol modifying HLC can not only reduce the polyer formed by the collagen due to the main hydrophobic bond of intermolecular interaction of HLC, but also prevent the aggregates formation of polymer in the purification process of HLC. So this new purification strategy breaks the difficulty in purifying the impurity and the aggregates formation owing to intermolecular interaction of HLC in the separation and purification process. The optimal condition of 1,2-propylene glycol as a modifier of HLC could not only gain the ideal separation effect but also achieve the efficient ultilization of chromatographic column through multiinjections. Meanwhile, triple helix structural characterization and morphology of the purified HLC were analyzed with Circular Dichroism (CD) spectroscopy, analytical gel-filtration chromatography and transmission electron microscope (TEM).2) Endotoxins of recombinant HLC is removed with the Pierce High-Capacity Endotoxin Removal resin. The difficult issue of recombinant protein produced in gene-engineering is the bacterial endotoxin contamination, which is one of the most difficult problems in recombinant protein at present. Therefore, we chose the High-Capacity Endotoxin Removal Resin which can adsorb more endotoxins and low cost in the endotoxins'removal of HLC. We had the description about the resin for reference, but its removal efficiency for endotoxin of HLC was not good. So in order to better remove endotoxins of HLC, the mild Tris-acetate (TA) buffer was used in this study. The conditions of TA buffer including ionic strength, pH, incubation time and HLC concentration were optimized for endotoxin of HLC. Avoiding HLC denatured and aggregates-forming is essential for obtaining the best effects for endotoxins'removal, at the same time, it must be ensured that other substances not introduced. This will achieve the high recovery of HLC and high efficientcy of endotoxins removal from HLC, and further provide the raw material for research and development of bioproduct.3) The regulation of phosphorylation activity of PRL-3 is studied in vitro.The phosphatase activity of phosphatase of regenerating liver phosphatases (PRLs) has been involved in variety of tumorgenesis and metastatic cancers, which has been shown to be associated with PRL-3 overexpression in a variety of cancers, but the regulation of the phosphatase activity of PRL-3 still remains unclear in the cancer metastasis.Therefore, we focused on the investigation of regulating phosphatase activity of PRL-3 in vitro. In the thesis, we mainly did study on the activity regulation of phosphorylation of PRL-3 phosphase and how to do it using phosphatases assays, potential substrates fishing for PRL-3 for kinase assay, peptides phosphorylation with MS-MS and Nuclear Magnetic Resonance (NMR) titration. This work can not only provide a target of cancer drug inhibitors, but also further support an evidance to explore phosphorylation mechanism of other PTPs.4) The interaction of the complex of PRL-3 and cystathionine ?-synthase (CBS) pair domains of cyclin M3 (CNNM3) is investigated by regulation of Cysteine phosphorylation. The phosphatases activity mechanism of PRLs can provide a potential evidence for cancer therapy, but PRL interacts with CNNM ion transporters and prevents CNNM depenent Mg2+ transport. Here, we focused on structural characterization of the complex of PRL-3 with CBS domain of CNNM3 using crystallization. The crystal structure of the complex of PRL-3 and CBS-pair domain of CNNM3 revealed the phosphorylation and binding sites. This is first identification of cysteine phosphorylation as a regulator protein modification, which is helpful for further exploring durg development, and detection and treatment incancer.5) The interaction between the treated HLC with high purity and PRL-3 are investigated by using cell experiments in vivo. This study investigated whether the treated HLC could suppresse PRL-3 overexpression with the wound-healing assays and western blotting assays for cell lysis. HLC will be provided for durg discovery and the therapy of cancer caused by the PRL-3 overexpression. Therefore, the exogenous supplement of HLC to body's collagen might reduce or inhibit PRL-3 overexpression which will cause the rapid metastasis of cancer.